The skeleton is one of the main targets of estrogen (E) action, as E regulates bone growth and remodeling. Although decreased E levels are known to be one of the main causes of osteoporosis, the specific molecular pathways by which E regulates bone metabolism are not fully characterized or understood. This Project uses a combination of novel mouse and cell models to define, at the cellular and molecular level, the signaling pathways by which E regulates bone turnover and bone mass, focusing on two modalities by which the estrogen receptor-alpha (ERa) functions: the classical mode of action, where ERa directly interacts with estrogen response elements (EREs) on DMA, and the non-classical mode, where ERa indirectly functions through protein-protein interactions with other transcription factors. Preliminary data demonstrates that mice containing an ERa mutation that eliminates classical ERa signaling (NERKI) exhibit osteopenia and impaired bone formation in both cortical and trabecular bone in males, but only in cortical bone in females. We hypothesize that due to increased E levels in female mice, ER(3 functions to mitigate the negative effects of the NERKI receptor in trabecular bone. In Aim 1, we directly test this by assessing whether loss of ERB leads to greater skeletal deficits in female, but not male NERKI mice. We will examine bones from wild type, NERKI//ERB+/+, and NERKI//ERB-/- mice using bone densitometry and histomorphometry. Aim 2 tests this hypothesis at the cellular level by assessing the ability of bone marrow stromal cells from these mice to commit and differentiate along the osteoblast lineage. Using quantitative polymerase chain reaction assays, we will test the hypotheses that expression of the NERKI receptor leads to impaired responses to Wnts and BMPs, and that ERP modulates these effects. Aim 3 examines loss of classical ERa signaling on the recruitment of the NERKI receptor to non-classical DNA binding sites using chromatin immunoprecipitation assays. Finally, Aim 4 uses a novel transgenic approach to examine the skeletal consequences of selective replacement of the endogenous ERa with the NERKI receptor only in osteoblasts. Collectively, these studies will provide a more detailed understanding of ER signaling pathways in bone.